Gluconeogenesis is a life-sustaining process for providing the sole fuel source for brain, testes and erythrocytes during starvation or prolonged fasting. Gluconeogenesis takes place mainly in liver in a metabolic pathway that is tightly regulated by insulin. When hepatic insulin signaling goes awry, gluconeogenesis becomes unabated, resulting in excessive glucose production and contributing to fasting hyperglycemia in diabetes. Our long-term goal is to characterize factors that link impaired insulin action to unrestrained gluconeogenesis. Our research identified FoxO6 as an important player in gluconeogenesis. FoxO6 is a new member of the forehead box O family, with unassigned function in metabolism. We show that hepatic FoxO6 activity is maintained at low basal levels in fed states, but is markedly unregulated in response to fasting. Augmented FoxO6 activity is detectable in insulin resistant livers, correlating with unrestrained gluconeogenesis in obesity and diabetes. FoxO6 stimulates gluconeogenesis in cultured hepatocytes and this effect is counteracted by insulin. Insulin inhibits FoxO6 activity via site-specific phosphorylation without altering its subcellular distribution, a distinct mechanism that distinguishes FoxO6 from other members of FoxO family. Our data underscore the importance of FoxO6 in glucose metabolism; spurring the hypothesis that FoxO6 dysregulation may contribute to the pathogenesis of fasting hyperglycemia in insulin resistant subjects. To address this hypothesis, we propose three specific aims: 1) To characterize the role of FoxO6 in gluconeogenesis and determine its contribution to blood glucose metabolism; 2) To investigate the distinct mechanism by which FoxO6 integrates insulin signaling to gluconeogenesis in liver; and 3) To determine the functional contribution of FoxO6 to the pathogenesis of fasting hyperglycemia in obesity and diabetes. To achieve these goals, we will employ gene transfer, transgenic overexpression, gene knockout and siRNA- mediated gene-silencing approaches to achieve FoxO6 gain- vs. loss-of-function in normal mice and mice with altered glucose metabolism. We have provided proof-of-principle and demonstrated the feasibility for the proposal. Accomplishing this project will deepen our understanding of insulin-dependent regulation of hepatic gluconeogenesis, by revealing a new regulatory pathway for fine-tuning the rate of hepatic glucose production between fasting and receding states. While the gluconeogenic pathway has been a major target for anti- hyperglycemia therapies, revelation of FoxO6-dependent gluconeogenic pathway will provide a potential therapeutic avenue for improving glycemic control in diabetes. PUBLIC HEALTH RELEVANCE: Excessive glucose production in liver is attributable to fasting hyperglycemia, a pathological condition that is commonly seen in subjects with morbid obesity or poorly controlled diabetes. The underlying mechanism is poorly understood. Our goal is to characterize factors that couple impaired insulin action to unrestrained glucose production for identifying novel therapeutic targets for improving glycemic control in diabetes.